The investigator's long-term goal is to understand how gene expression is regulated in Chlamydia with the aim of identifying therapeutic targets for interrupting the chlamydial life cycle. The general strategy is to characterize transcription initiation in vitro, as methods for stable gene transfer are not available in Chlamydia at this time. The objective of this application is to characterize basal and regulated promoter activity. The central hypothesis is that transcription in Chlamydia is regulated by activators and repressors, and by alternative sigma factors that transcribe different classes of promoters. Three specific aims are proposed: 1. Characterization of C. trachomatis promoter structure; It is hypothesized that many chlamydial promoters do not have an optimal promoter structure and require activation. The dnaK and groE promoters will be defined with a mutational approach that uses in vitro transcription to assay for loss or gain of function. Promoter structure will be independently defined with physical methods, The Spacer A/T region and the UP element are cis-acting sequences that enhance transcription in Chlamydia. To ascertain if either has a general role in promoter activation, candidate Spacer A/T regions and UP elements in selected chlamydial promoters will be examined to determine if mutations in these regions have an effect on in vitro transcription. 2. Investigation of the role of the Spacer A/T region. It is hypothesized that the Spacer A/T region regulates promoter activity by functioning as a binding site for a putative activator. To test this hypothesis, different RNAP preparations will be compared to determine if activation via the Spacer A/T region can be separated from basal promoter activity. Heterologous activation of E. coli RNAP transcription will also be tested for possible exploitation in a future genetic approach to clone the activator. Physical methods will be used to determine if the Spacer A/T region makes a contact with the activator. 3. Reconstitution of regulated promoter activity in vitro. The hypothesis that promoter activity is regulated during the developmental life cycle and in response to environmental stimuli will be tested by comparing reconstituted RNAP activities for promoter-specific effects on transcription. RNAP activity will be reconstituted by using RNAP extracts prepared under different conditions, cell extracts, purification fractions and selected purified recombinant proteins. Two putative alternative sigma factors will be studied to determine their roles in regulating chlamydial transcription.